Engineered living materials for sustainable and green applications

This PhD thesis explores the development of engineered living materials (ELMs) with applications in biosensing, biomanufacturing, and environmental sustainability. Combining synthetic biology, bioprinting, and materials science, this research focuses on creating functional bacterial systems capable of operating in real-world conditions, with potential use in diagnostics, agricultural monitoring, and carbon capture technologies. In the first section, a robust bioprinting workflow was developed to fabricate bacterial biosensors. These biosensors were tested in a range of environments, including nutrient-poor water samples and complex clinical matrices like culture supernatants and bronchial aspirates. The devices successfully detected target molecules, such as bacterial autoinducers from Vibrio fischeri and Pseudomonas aeruginosa, even under challenging conditions. The bioprinted materials maintained viability and functional stability for over two weeks, with the biosensors being storable for up to one month under refrigeration. Additionally, incorporating multiple bacterial strains into the biosensors enabled multiplexed sensing and cell-cell communication, significantly enhancing the complexity and utility of the devices. This work laid the foundation for automating bioprinting processes and designing bacterial-laden biosensors for environmental monitoring and clinical applications. The second phase of the thesis focuses on agricultural and industrial applications, particularly in developing biosensors to detect volatile organic compounds (VOCs), acetate, nitrates, and phosphates. These biosensors were envisioned as tools to improve sustainable fertilization practices by monitoring nutrient levels in soil, thus reducing environmental impacts and enhancing crop production, or to optimize biogas processes. Eleven biosensors were characterized, and promising candidates were identified for detecting the target molecules. However, factors like pH and the presence of complex substances in soil and digestate samples affected sensor performance, highlighting the need for extensive optimization to ensure reliable biosensing in field applications. In the final section, cyanobacteria were explored as a platform for biomanufacturing and ELMs aimed at climate change mitigation. The fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 was engineered to either surface-display or secrete proteins and enzymes for a range of applications, including therapeutics and carbon-negative practices. A novel S-layer protein was characterized, enabling the secretion and display of industrially relevant enzymes. Furthermore, cyanobacteria-based living bricks capable of CO₂ fixation, biocement production, and self-repair were developed. These living bricks demonstrated promising mechanical properties, but challenges such as uneven cell distribution and variability in biomineralization efficiency remain to be addressed. The research also explored the use of engineered carbonic anhydrase enzymes for improving CO₂ capture, with several variants showing potential for enhancing carbonate precipitation. Finally, strategies to improve cyanobacterial resilience in harsh environments, such as desiccation, were investigated by leveraging the expression of tardigrade-specific intrinsically disordered proteins (TDPs), further supporting the use of cyanobacteria in challenging environmental conditions. In conclusion, this thesis demonstrates the potential of ELMs for biosensing, biomanufacturing, and environmental sustainability, offering promising avenues for the development of living technologies that can address some of the most pressing issues facing society today.